Widely used general anesthetics require administration by highly trained and vigilant experts, in part because they produce many undesirable effects and can be lethal at clinically used doses, particularly in elderly patients an those with systemic diseases. The safest (highest animal LD50/ED50 ratios) anesthetics, etomidate and alphaxalone, are both potent and selective modulators of GABAA receptors that also display stereoselectivity. These and similar drugs represent optimal starting points for developing improved and safer anesthetics. The overall aim of this renewal proposal is to define the interactions of etomidate, alphaxalone, propofol (a potent but less safe anesthetic) and a novel potent barbiturate (mTFD-MPAB) at their sites on pentameric (?1)2(?3)2?2L GABAA receptors, and to clarify conformational changes in these sites that couple anesthetic binding to functional effects. Our working hypothesis is that these drugs act at distinct sites within the fiv inter-subunit transmembrane clefts formed by M1, M2, and M3 domains, and that these clefts expand during receptor activation and desensitization. Our studies to date have identified novel contact points for etomidate, propofol, alphaxalone, and mTFD-MPAB within multiple inter-subunit clefts. We also have engineered cysteines into these sites to reveal GABA-enhanced sulfhydryl modification and GABA-dependent anesthetic protection. We have also developed quantitative allosteric models that explain multiple GABAAR mutant functional phenotypes, including subsite contributions to each drug's actions.
In Aim 1, we will identify which of the fiv inter-subunit sites interacts with each anesthetic, and which subsites transduce drug binding into enhanced receptor gating. We will use cysteine-substitutions in transmembrane domains M1, M2, and M3 of each subunit, along with chemical modification, protection, and electrophysiology, to define the binding clefts and residues closest to each anesthetic.
Aim 2 will test the hypothesis that GABA activation enlarges all five inter-subunit transmembrane clefts in concert. We will examine anesthetic subsite interactions using cysteine accessibility assays and estimate the effective "cut-off" size of the anesthetic binding pockets using variable size mutations and cysteine adducts for three anesthetics that bind in the same inter-subunit clefts. We will also use novel sulfhydryl-modifying anesthetic derivatives and cysteine substituted residues to further define the binding orientation of stereoselective anesthetics.
Aim 3 will test whether anesthetics influence GABAA receptor rates of desensitization and whether the shape of the inter-subunit clefts differs between open and desensitized states, again based on cysteine accessibility. These studies will use submillisecond solution switching and patch-clamp electrophysiology. Data will be analyzed using functional allosteric models and further interpreted in the context of evolving GABAAR structural models.
General anesthetics are among the most beneficial but also the most dangerous drugs in routine clinical use. To guide development of future anesthetics, we aim to understanding the mechanisms of anesthetics proven safest in animals, which are potent and stereoselective modulators of GABAA receptors: etomidate, alphaxalone, and a novel barbiturate. We will use electrophysiology, targeted cysteine substitutions, chemical modification, and protection assays to map the transmembrane inter-subunit GABAA receptor sites for these anesthetics, to determine the nature of their rearrangements during both channel activation and desensitization, and to establish the orientation of stereoselective anesthetic binding in these sites.
|Forman, Stuart A; Chiara, David C; Miller, Keith W (2015) Anesthetics target interfacial transmembrane sites in nicotinic acetylcholine receptors. Neuropharmacology 96:169-77|
|Feng, H-J; Jounaidi, Y; Haburcak, M et al. (2014) Etomidate produces similar allosteric modulation in *1*3ýý and *1*3ýý2L GABA(A) receptors. Br J Pharmacol 171:789-98|
|Stewart, Deirdre S; Pierce, David W; Hotta, Mayo et al. (2014) Mutations at beta N265 in ?-aminobutyric acid type A receptors alter both binding affinity and efficacy of potent anesthetics. PLoS One 9:e111470|
|Stewart, Deirdre S; Hotta, Mayo; Desai, Rooma et al. (2013) State-dependent etomidate occupancy of its allosteric agonist sites measured in a cysteine-substituted GABAA receptor. Mol Pharmacol 83:1200-8|
|Forman, Stuart A (2013) A paradigm shift from biophysical to neurobiological: the fading influence of Claude Bernard's ideas about general anesthesia. Anesthesiology 118:984-5|
|Stewart, Deirdre S; Hotta, Mayo; Li, Guo-Dong et al. (2013) Cysteine substitutions define etomidate binding and gating linkages in the *-M1 domain of ýý-aminobutyric acid type A (GABAA) receptors. J Biol Chem 288:30373-86|
|Forman, Stuart A (2013) The expanding genetic toolkit for exploring mechanisms of general anesthesia. Anesthesiology 118:769-71|
|Guitchounts, Grigori; Stewart, Deirdre S; Forman, Stuart A (2012) Two etomidate sites in ýý1ýý2ýý2 ýý-aminobutyric acid type A receptors contribute equally and noncooperatively to modulation of channel gating. Anesthesiology 116:1235-44|
|Forman, Stuart A; Stewart, Deirdre (2012) Mutations in the GABAA receptor that mimic the allosteric ligand etomidate. Methods Mol Biol 796:317-33|
|Ruesch, Dirk; Neumann, Elena; Wulf, Hinnerk et al. (2012) An allosteric coagonist model for propofol effects on ýý1ýý2ýý2L ýý-aminobutyric acid type A receptors. Anesthesiology 116:47-55|
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